This project defines the biochemical structure and function of receptors, transduction proteins and the NADPH oxidase effector system involved in human neutrophil and monocyte responses to inflammation and infection. Studies focus upon: (i) chemotactic receptors for formyl peptides (FPCR), C5a, platelet activating factor (PAF) and ATP (P2); and (ii) NADPH oxidase components in the cytosol p47-phox, p67-phox, NCF-3 and the membrane cytochrome b558 (CYTO b) subunits P22-phox and gp9l-phox mediating microbicidal superoxide production. Using myeloid cell mRNA in a frog oocyte expression system we demonstrate chloride currents and calcium effluxes in response to FMLP, C5a, PAF, and ATP. These responses are mediated by mRNA transcripts of 2 kb for FMLP and C5a receptors and 4 and 6 kb for PAF and ATP. FMLP, C5a and ATP responses are pertussis toxin sensitive (mediated by G-protein) while PAF responses are not. We are the first to delineate and clone cDNA encoding two neutrophil cytosol factors p47-phox and p67-phox essential to NADPH oxidase activation. Both proteins contain a pair of homologous sequences with similarity to the src-oncogene SH3 (A-box) regulatory region. We show that p47-phox undergoes a series of phosphorylations culminating in the interaction of p47 with a distinct amino acid sequence (RGVHFIF) present in the cytoplasmic domain of cytochrome b558 gp9l subunit. This is followed by translocation of other cytoplasmic oxidase components to the membrane and activation of the oxidase. Using a baculovirus expression system, active recombinant p47-phox and p67-phox have been used to demonstrate the absolute requirement for a third cytoplasmic factor, NCF-3, for activation of the oxidase.
